HIV/AIDS is a major cause of global morbidity and mortality. Direct killing of infected CD4+ T cells, indirect killing of bystander immune cells and evasion of immune responses all underlie the pathogenesis of severe immune dysfunction caused by HIV/AIDS. Viral proteins that mediate immune dysfunction and evasion include the membrane-associated myristoylated Nef protein, which is also critical for optimal viral replication and increased virulence. Nef mechanisms of immune evasion have not been fully delineated, but include downregulation of major immune cell receptors CD4 and MHC class I. CD4 is the major T cell receptor for HIV and is critical for helping both cell mediated and humoral immunity. CD4 downregulation therefore leads to severe immune dysfunction. MHC class I receptors are critical for presentation of viral antigens on infected cells that are then recognized by CD8 killer cells. Loss of MHC-I density at the cell surface will allow HIV infected cells to escape immune-surveillance. [unreadable] Genetic and biochemical studies have suggested two different mechanisms for the CD4 and HLA-I downregulation induced by Nef. Numerous studies have suggested that Nef enhances endocytic clearance of immune cell receptors by clathrin coated vesicles that use a specific class of AP2 coat proteins. However, other mechanisms have also been proposed for Nef induced CD4 and MHC-I defects are not fully understood. This project has focused on identifying the precise mechanisms.[unreadable] In the past reporting period, we showed previously that [DE]XXXL[LI]-type peptide signals from HIV Nef interacted in a bipartite manner with combinations of gamma-sigma1 of AP-1 and delta-sigma3 subunit heterodimers of the AP-1 and AP-3 vesicles respectively. In accordance, Nef mutants altered at the [DE]XXXL[LI]- motif were severely defective for CD4 downregulation. [unreadable] During the current reporting period, we have extended the above studies to show that the loss of CD4 at the cell surface in HIV-1 Nef expressing cells did not result exclusively from accelerated endocytosis of CD4. Both the recycling cell surface receptor and the nascent receptor in transit to the plasma membrane were susceptible to intra-cellular retention and degradation by HIV-1 Nef. By using genetic inhibitors of endocytosis and siRNA induced knock down of AP-2, we confirmed that accelerated CD4 endocytosis was not a dominant mechanism of HIV-1 Nef. In contrast, SIV Nef, which has a canonical ?tyrosine? motif near the N-terminus that binds to the AP-2 medium (mu) subunit. AP-2 mediated enhanced endocytosis constituted the predominant mechanism for SIV Nef induced downregulation of CD4. HIV-1 Nef which lacks the tyrosine motif and mu-2 binding potential, enhanced CD4 endocytosis only slightly. However, both HIV-1 and SIV Nef have a [DE]XXXL[LI] motif and trap both the nascent CD4 en route to plasma membrane and the internalized CD4 during recycling, presumably through a tripartite recognition between the signal in Nef and the C-tail of CD4 with the two subunits of the AP-1 and AP-3 complexes. Thus, HIV Nef proteins have evolved preferential usage of dileucine based protein sorting mechanisms.[unreadable] Human chemokine and chemokine receptor dysfunction are associated with numerous acute and chronic diseases such as asthma and arthritis and infectious diseases including AIDS and TB. HIV infection induces severe defects in lymphocyte homing, DC and macrophage maturation and TH1/TH2 polarization of lymphocytes. HIV infected lymphocytes and monocytes display aberrant cytokine and chemokine secretion potential. And some members of the human chemokine receptor family serve as coreceptors for HIV entry besides their essential roles in regulating leukocyte migration. Our understanding of this feedback controlled signaling system in health and disease will enable us to understand viral pathogenesis and help us develop novel therapeutic interventions.[unreadable] Chemokine receptors constitute a sub-class in the GPCR superfamily, which is phylogenetically conserved from yeast to primates and regulate diverse biological processes including the mating response of yeast, angiogenesis, leukocyte chemotaxis in inflammation and disease, and sensory perception in higher eukaryotes. Analogous to other GPCRs, ligand binding to the chemokine receptors induces conformational change that recruits G-alpha subunit of trimeric G protein followed by GTP hydrolysis. This sets up a cascade of events leading to polarized cellular motility and other activation pathways. [unreadable] In previous years a) we identified the structural requirements of CC and CXC chemokine receptors for the biological function and HIV usage; b) investigated how naturally occurring CCR5 mutants impaired for surface expression impact on the physiological function of the wt receptor and its use as a HIV coreceptor; c) addressed the mechanistic differences between the CCR5 and CXCR4 receptors in the agonist-driven receptor signaling, desensitization and internalization. This work identified a need for a more precise and exact knowledge of normal chemokine receptor signaling pathways in primary leukocytes. Along these lines, we showed in the previous reporting period that in neutrophils, chemokine mediated cell migration and receptor internalization are separable processes. Rather than being integral to the process of cell migration, receptor endocytosis is a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest